Interactive variable pathlength device

ABSTRACT

This disclosure relates generally to a sampling device, and more particularly, a sampling device that facilitates spectroscopic measurements with a variable path length and the necessary software controlled algorithms and methods for such a device.

The present application is a continuation of U.S. patent applicationSer. No. 13/199,835 filed Sep. 9, 2011 which is a continuation of U.S.patent application Ser. No. 12/800,860 filed May 24, 2010 which is acontinuation of U.S. patent application Ser. No. 12/100,467 filed Apr.10, 2008 which claims benefit under 35 USC §119(e) of the U.S.Provisional patent Application Ser. No. 60/923,179 filed Apr. 13, 2007.

FIELD OF THE INVENTION

The present invention relates generally to a sampling device, and, moreparticularly, a sampling device that facilitates spectroscopicmeasurements with a variable path length and the necessary softwarecontrolled algorithms and methods for using such a device.

BACKGROUND OF THE INVENTION

Spectroscopic analysis is a broad field in which the composition andproperties of a material in any phase, gas, liquid, solid, aredetermined from the electromagnetic spectra arising from the interaction(eg. absorption, luminescence, or emission) with energy. One aspect ofspectrochemical analysis, known as spectroscopy, involves interaction ofradiant energy with the material of interest. The particular methodsused to study such matter-radiation interactions define many sub-fieldsof spectroscopy. One field in particular is known as absorptionspectroscopy, in which the optical absorption spectra of liquidsubstances are measured. The absorption spectra is the distribution oflight attenuation (due to absorbance) as a function of light wavelength.In a simple spectrophotometer the sample substance which is to bestudied is placed in a transparent container, also known as a cuvette orsample cell. Electromagnetic radiation (light) of a known wavelength, λ,(ie. ultraviolet, infrared, visible, etc.) and intensity I is incidenton one side of the cuvette. A detector, which measures the intensity ofthe exiting light, I is placed on the opposite side of the cuvette. Thelength that the light propagates through the sample is the distance d.Most standard UV/visible spectrophotometers utilize standard cuvetteswhich have 1 cm path lengths and normally hold 50 to 2000 μL of sample.For a sample consisting of a single homogeneous substance with aconcentration c, the light transmitted through the sample will follow arelationship know as Beer's Law: A=εcl where A is the absorbance (alsoknown as the optical density (OD) of the sample at wavelength λ whereOD=the −log of the ratio of transmitted light to the incident light), εis the absorptivity or extinction coefficient (normally at constant at agiven wavelength), c is the concentration of the sample and l is thepath length of light through the sample.

Spectroscopic measurements of solutions are widely used in variousfields. Often the compound of interest in solution is highlyconcentrated. For example, certain biological samples, such as proteins,DNA or RNA are often isolated in concentrations that fall outside thelinear range of the spectrophotometer when absorbance is measured.Therefore, dilution of the sample is often required to measure anabsorbance value that falls within the linear range of the instrument.Frequently multiple dilutions of the sample are required which leads toboth dilution errors and the removal of the sample diluted for anydownstream application. It is, therefore, desirable to take existingsamples with no knowledge of the possible concentration and measure theabsorption of these samples without dilution.

Multiple sample cuvettes may solve the problem of repetitive sampling,however, this approach still requires the preparation of multiple samplecuvettes and removes some sample from further use. Furthermore, in mostspectrophotometers the path length, l, is fixed.

Another approach to the dilution problem is to reduce the path length inmaking the absorbance measurement. By reducing the measurement pathlength, the sample volume can be reduced. Reduction of the path lengthalso decreases the measured absorption proportionally to the path lengthdecrease. For example, a reduction of path length from the standard 1 cmto a path length of 0.2 mm provides a virtual fifty-fold dilution.Therefore, the absorbance of more highly concentrated samples can bemeasure within the linear range of the instrument if the path length ofthe light travelling through the sample is decreased. There are severalcompanies that manufacture cuvettes that while maintaining the 1 cm²dimension of standard cuvettes decrease the path length through thesample by decreasing the interior volume. By decreasing the interiorvolume less sample is required and a more concentrated sample can bemeasured within the linear range of most standard spectrophotometers.While these low volume cuvettes enable the measurement of moreconcentrated samples the path length within these cuvettes is stillfixed. If the sample concentration falls outside the linear range of thespectrophotometer the sample still may need to be diluted or anothercuvette with an even smaller path length may be required before anaccurate absorbance reading can be made.

The prior art also describes spectrophotometers and flow cells that arecapable of measuring absorbance values of low volume samples. Thesedevices are designed to utilize short path lengths for measuringabsorbance so that only small amounts of sample are required. U.S. Pat.No. 4,643,580 to Gross et al. discloses a photometer head in which thereis a housing for receiving and supporting small test volumes. A fiberoptic transmitter and receiver are spaced within the housing so that adrop can be suspended between two ends.

U.S. Pat. No. 4,910,402 to McMillan discloses an apparatus in which asyringe drops liquid into the gap between two fixed fibers and an IRpulse from an LED laser is fed through the droplet. The output signal isanalyzed as a function of the interaction of the radiation with theliquid of the drop.

U.S. Pat. No. 6,628,382 to Robertson describes an apparatus forperforming spectrophotometric measurements on extremely small liquidsamples in which a drop is held between two opposing surfaces by surfacetension. The two surfaces can move relative to one another to keep thesurface tension in a sample such that a spectrophotometric measurementby optical fibers can be made.

U.S. Pat. No. 6,747,740 to Leveille et al. describes a photometricmeasurement flow cell having measurement path lengths that can beadjusted down to less than 0.1 mm. The flow cell contains a steppedoptical element which includes a stem portion that can be made tovarious lengths. The measurement path length can be adjusted byreplacing one of the stepped elements of a particular length withanother stepped element of a different length.

U.S. Pat. No. 6,188,474 to Dussault et al. describes a sample cell foruse in spectroscopy that included two adjustable plates that enable auser to vary the cross sectional geometry of a sample cell flow pathbetween two or more configurations.

U.S. Pat. No. 6,091,490 to Stellman et al. describes a fiber opticpipette coupled to a glass capillary for spectrophotometric measurementsof small volume samples utilizing long path length capillaryspectroscopy.

There are a series of patents assigned to Molecular Devices Corporationthat describe a microplate reader capable of determining absorptionmeasurements for multiple liquid samples in microtiter plates. Each wellof the microtiter plate may provide for a different light path lengthbased on the amount of sample solution in each well and the curvature ofthe meniscus of the solution in each well.

While some of these instruments provide the capability of varying thepath length for measurement of highly concentrated low volume samplesthe applications described therein relate primarily to single pathlength and single wavelength measurements. Several of the instrumentsprovide a limited number of path lengths and all are limited to pathlength larger than 0.2 mm. Furthermore, the devices and methods of theprior art do not provide for expanding the dynamic range of thespectrophotometer so that it is not necessary to adjust theconcentration of the sample to fall within the linear range ofabsorbance detection of the instrument. To the extent that the prior artteaches shorter path lengths to determine the concentration of veryconcentrated samples or low volume samples the focus of these devices isto take a single absorbance reading at a single path length. As such theprior art references require that the path length be known with greataccuracy so that an accurate concentration measurement can be made.

The present invention provides devices and methods that provide avariable path length spectrophotometer which dynamically adaptsparameters in response to real time measurements via software control toexpand the dynamic range of a conventionally spectrophotometer such thatsamples of almost any concentration can be measured without dilution orconcentration of the original sample. Furthermore, certain methods ofthe present invention do not require that the path length be known todetermine the concentration of samples. This and other objects andadvantages of the invention, as well as additional inventive features,will be apparent from the description of the invention provided herein.

SUMMARY OF THE INVENTION

The present invention overcomes the disadvantages and shortcomings ofthe prior art by providing an interactive variable path length devicesand methods for spectroscopic measurement of a sample. The instrumentsof the present invention can be used to measure the concentration ofvery concentrated samples by providing path lengths around 0.2 μm andabove. Such small path lengths permit the measurement of samples tooconcentrated to be measured by conventional spectrophotometers.Furthermore, the instruments and methods of the present invention canprovide spectrum scans in two or three different path length zones. Thisenables users to determine optimal absorbance peaks in a sample in asingle run. The benefit of this method is that it can provideinformation on optimization of concentration measurements by comparingabsorbance peak data at multiple path lengths and multiple wavelengthsas these values can be different due to the contents in the sample.Instruments that use standard fixed path length cuvettes can not presentall of this data at the same time. The variable path length instrumentmay include a probe tip, sample vessel, a mechanism for moving the probetip and sample vessel relative to one another (eg. the sample vessel isstationary and the probe moves or the probe is stationary and the samplevessel moves or both are capable of movement), delivery optical fiber,detector and appropriate software for path length control andmeasurement parameters.

The present invention includes methods of determining the concentrationof a sample comprising placing the sample in a vessel; moving a proberelative to the vessel such that the probe makes contact with the bottomof the vessel; moving the probe relative to the vessel such that theprobe moves from the bottom of the vessel through the sample by apredetermined increment such that a preselected path length through thesolution is obtained; taking an absorbance reading at a predeterminedwavelength; repeating steps of moving the probe relative to the sampleand taking a measurement; generating a regression line from theabsorbance and path length such that a slope of the regression line isobtained; determining the concentration of the sample by dividing theslope of the regression line by the extinction coefficient of thesample.

The present invention also includes instruments for determining theconcentration of a sample at multiple path lengths comprising a lightsource operably linked to a probe; a sample vessel that can contain thesample; a motor operably linked to the sample vessel such that thesample vessel can be moved relative to the probe to provide variablepath lengths; a probe that can carry electromagnetic radiation that canbe moved relative to the sample vessel by the motor; a detector that candetect electromagnetic radiation disposed such that the detector issubstantially perpendicular to the electromagnetic radiation emanatingfrom the probe; and software that can calculate the concentration of thesample based on the information provided by the detector at thepredetermined path length.

The instruments and methods of the present invention can be used inconjunction with a standard spectrophotometer which may be used toprovide an electromagnetic source and/or a detector for measuringelectromagnetic radiation.

FIGURES

FIG. 1 is a flow diagram of one possible embodiment of the variable pathlength device software set up.

FIGS. 2A and 2B are flow diagrams of the data acquisition of thevariable path length instrument software.

FIG. 3 is a flow diagram of the data acquisition of the variable pathlength instrument software

FIG. 4A is a schematic of one embodiment of the instrument of thepresent invention.

FIG. 4B is a schematic of one embodiment of the probe tip assembly.

FIG. 5 is a schematic of a flow-through device which may serve as asample vessel in the instruments of the present invention.

FIG. 6 shows the spectra of stock and diluted CSA from Cary400 andSoloVPE taken at a 1 mm and 10 mm path length

FIG. 7 shows the regression line of a plot of Absorbance at 285 nmversus path length for a stock solution of CSA.

FIG. 8 shows the regression line of a plot of Absorbance at 285 nmversus path length for a diluted solution of CSA.

FIG. 9 is spectra of Patent Blue Standard at path lengths from 15.0 mmto 1.0 mm.

FIG. 10 is spectra of Patent Blue Standard at path lengths from 1.5 mmto 0.1 mm.

FIG. 11 is the spectra of BSA from 200 to 340 nm at 10 mm and 1 mm pathlength on a standard spectrophotometer.

FIG. 12 is the spectra of BSA from 200 to 340 nm at 200 μm path length

FIG. 13 is the spectra of BSA from 200 to 340 nm at multiple pathlengths between 0.0.1 mm and 0.1 mm on an instrument of the presentinvention

FIG. 14 is a plot of a linear regression line for the plot of theabsorbance versus path length for BSA at 280 nm.

DEFINITIONS

The term “moving the probe relative to the vessel” or “moving the proberelative to the sample” means that the vessel or the sample relative tothe probe is moved. This encompasses the situations where the probe ismoving and the vessel or sample is stationary, the vessel or sample ismoving and the probe is stationary and where the sample or the vessel ismoving and the probe is moving.

The term “taking an absorbance reading” means that any absorbancereading(s) is measured by the device or instrument. This encompassessituations where the absorbance reading is taken at a single wavelengthand/or a single path length or where the reading is taken at multiplewavelengths (such as in a scan) and/or multiple path lengths.

The term “sample(s)” may include, but is not limited to, compounds,mixtures, surfaces, solutions, emulsions, suspensions, cell cultures,fermentation cultures, cells, tissues, secretions, and extracts.

The term “motor” is any device that can be controlled to provide avariable path length through a sample.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to devices and methods for determining thespectrophotometric characteristics of a solution by employing anapproach that permits the use of a variable path length for multipledeterminations of the parameters of interest. For example, indetermining the concentration of a compound in solution the presentinvention provides methods and devices for determining the absorbance ofthe solution at various path lengths. The values of the absorbance atvarious path lengths can then be used to calculate the concentration ofthe compound in the solution. The devices and methods of the presentinvention are particularly useful for determining the concentration ofhighly concentrated samples without resorting to single or multipledilutions of the samples. This attribute is possible due to the smallpath lengths which the devices of the present invention can achieve. Theinstruments of the present invention can be used to measure theconcentration of very concentrated samples by providing path lengthsaround 0.2 μm and longer. Preferably the instruments of the presentinvention can provide path lengths from about 0.5 μm and to about 15 cmand more preferably between about 1 μm to about 50 mm. The devices andmethods also provide for measurement of concentrations of extremelydilute solutions by providing larger path lengths. In essence thedevices and methods of the present invention expand the dynamic range ofa standard spectrophotometer by permitting a wide range of path lengthsfor measuring the absorbance values of a solution. This broad dynamicrange enables users to determine the concentrations of their sampleswithout altering (diluting or concentrating) the samples. Whilepreferred embodiments of the methods and devices of the presentinvention are for determining the absorbance, extinction coefficient orconcentration of a particular sample or set of samples the devices andmethods of the present invention may also be used in different modessuch as scattering, luminescence, photoluminescence, photoluminescencepolarization, time-resolved photoluminescence, photoluminescencelife-times and chemiluminescence as well as other modalities. Thedevices and the methods of the present invention may be used todetermine optical values of one or more samples at a given time. Theinvention contemplate the use of single sample formats such as cuvettesor any sample holder, as well as multiple sample formats such asmicrotiter plates and multiple cuvette or multiple sample arrangements.

The variable path length device of the present invention may becomprised of a probe tip, sample vessel, motor, delivery optical fiber,detector, unidirectional sliding mechanism and appropriate software forpath length control and measurement parameters.

Probe Tip

In the present invention the probe tip is a light delivery device whichdelivers light to the sample.

The probe tip may be a single light delivery device such as a fiberoptic cable that interfaces with one or more electromagnetic sources topermit passage of light through the sample. Alternatively the probe tipmay be housed in a probe tip assembly which may be comprised of a lightdelivery device, housing, end terminations and other optical componentsand coatings. The light delivery device can be fused silica, glass,plastic or any transmissible material appropriate for the wavelengthrange of the electromagnetic source and detector. The light deliverydevice may be comprised of a single fiber or of multiple fibers andthese fibers can be of different diameters depending on the utilizationof the instrument. The fibers can be of almost any diameter but in mostembodiments the fiber diameter is in the range of from about 0.005 mm toabout 20.0 mm. In a preferred embodiment the light delivery device is asingle optical fiber with a diameter of from about 0.1 mm to about 1.0mm. The probe tip optionally utilizes a housing to contain the lightdelivery device. This housing is used primarily to shield the lightdelivery device and may be made from metal, plastic, ceramic or anyother material that is compatible with its usage. The probe tip mayoptionally include end terminations such as connectors, ferrules oranything that will facilitate a mechanical interconnection. Theterminations can be polished, cleaved, shaped or manipulated in anyfashion compatible with the device's usage. The instruments of thepresent invention include probe tips with additional optical componentssuch as lenses or filters. The probe tips may include coatings on theend of the fiber tip to serve as filters, pH indicators, catalysts or assealing mechanisms. The probe tip may be a permanent part of theinstrument and/or probe assembly device or alternatively the probe tipmay be detachable, such that it may be removed from the probe tipassembly. As a permanent part of the instrument the probe tip is anintegral part of the light delivery device. In a preferred embodimentthe probe tip is a single optical fiber which is attached at one end tothe light source and at the other end immersed in the sample.Alternatively the probe tip may be detachable and in such embodimentsthe probe tip can be separated from the light delivery device though avariety of mechanisms. In a preferred embodiment the probe tip isattached to the light delivery device though a Touhey Borst adapter suchthat after usage the probe tip can be removed and replaced with anotherprobe tip. The detachable probe tip is of a length sufficient topenetrate the sample and attach to the light delivery assembly. Inpreferred embodiments of the detachable probe tip the length of theprobe tip is at least about 20 mm in length. Depending on its usage theprobe tip may simply be thrown away after removal. Disposable probe tipsobviate problems associated with cleaning the probe tip and avoid thepotential of contamination from one sample to another. Instruments ofthe present invention include multiple probe tips that can be associatedwith a single light delivery device. Alternatively multiple lightdelivery devices may be associated with each probe tip.

The path length is the distance between the end of the probe tip andinside surface of the sample vessel holding the liquid, the insidesurface being the surface of the vessel which is substantiallyperpendicular to the probe tip. The end surface of the probe tip, whichboth defines the path length and is in contact with the liquid, issubstantially parallel to the inside surface of the sample vessel whichis adjacent to the detector. In one embodiment, the probe tip ispositioned above the sample vessel holding the sample and aligned sothat the light exiting the probe tip will pass through the sample vesselonto a detector (or detection light guide). The probe tip is able totransmit wavelengths within the range of the instrument.

Light Source

The electromagnetic radiation source provides light in a predeterminedfashion across a wide spectral range or in a narrow band. The lightsource may include arc lamps, incandescent lamps, fluorescent lamps,electroluminencent devices, laser, laser diodes, and light emittingdiodes, as well as other sources. In a preferred embodiment the sourceof radiation is a Xenon arc lamp or tungsten lamp. In a preferredembodiment of the present invention the light source is coupled to theprobe tip through a light guide. Alternatively the light source could bea light emitting diode that can be mounted directly onto the probe tip.

Sample Vessel

The vessel must be able to contain the liquid and allow light to passthrough it onto the detection light guide or detector. The vessel willalso have an opening to allow the probe tip to delivering light, topenetrate the liquid. This vessel should be able to transmit wavelengthswithin the range of the instrument typically from about 200-1100 nm. Forultraviolet application a quartz vessel may be required, but oftenplastic vessels will made of cyclo olefin polymer (COP), cyclo olefincopolymer (COC), polystyrene (PS) or polymethyl methacrylate (PMMA) willsuffice. The sample vessels used with the present invention can be ofdifferent sizes and shapes depending upon the application and the amountof sample available for analysis. The sample vessels of the presentinvention may be anything that permits an absorbance value to be taken.Such vessels include stationary sample vessels as a cuvette ormicrotiter plate or moving samples as in a flow-through device (FIG. 5).The sample size may be between 0.1 μL to several liters in a stationarysample. The preferred shape of the vessel is one with the side facingthe detector being substantially flat and substantially parallel to theface of the detector. The detector may be situated at a slight angle tothe vessel to reduce noise due to back reflection of the electromagneticradiation coming through the sample. The sample vessel may have multiplewells such as in a microtiter plate. The sample vessel may be coatedwith optical materials or chemicals or biochemicals such as antibodies.The sample vessel may optionally be heated or cooled by the instrumentand may be held in a sealed area that can be sterile or non-sterile. Thesample may be held in a sample holder supported by a stage. The samplecan include compounds, mixtures, surfaces, solutions, emulsions,suspensions, cell cultures, fermentation cultures, cells, tissues,secretions, extracts, etc. Analysis of the sample may involve measuringthe presence, concentration or physical properties of a photoactiveanalyte in such a composition. Samples may refer to contents of a singlewell or cuvette or sample holder or may refer to multiple samples withina microtiter plate. In some embodiments the stage may be outside theinstrument.

Motor

The motor drives the tip probe into and out of the vessel. The motordrives the probe tip in precise steps to vary the path length throughthe sample. Path length changes can be from zero mm and larger dependingupon device configuration. The motor permits the movement of the probewithin the sample to place the probe tip at the precise pre-determinedpath length. Motors that can be used with the instruments of the presentinvention include stepper motors, servo, piezo, electric and magneticmotors or any device that can be controlled to provide a variable pathlength through a sample. In a preferred embodiment of the instruments ofthe present invention the motor drives a stage on which the samplevessel rests so that the probe tip moves relative to the sample vessel.In this configuration the stage and the probe move relative to eachother in increments which range from 0.2 μm to 1 cm. In a preferredembodiment the range of increment is between from about 1 μm to about 50μm. The relative motion of the stage to the probe is accurate to with aresolution of 0.2 μm or less. In a preferred embodiment of theinstruments of the invention the resolution of the relative motion ofthe probe and the stage is between about 0.5 μm to about 0.01 μm.

Unidirectional Sliding Mechanism

The unidirectional sliding mechanism is a system designed to permitphysical contact between the end of the probe tip and the “bottom”(perpendicular to the probe tip) of the sample vessel in order toestablish a “zero path length” position which is an approximate zerobenchmark from which all other path lengths can be referenced. In apreferred embodiment of the present invention the unidirectional slidingmechanism insures that the probe tip makes physical contact with thesample vessel surface thereby guaranteeing that the probe tip is in the“zero path length” position. Physical contact should to be achievedwithout causing damage to either the sample vessel or the probe tip. Ina preferred embodiment the position is achieved by allowing/requiringlinear displacement of either the sample vessel of the probe tip in onedirection once the physical contact is achieved. This allowsdisplacement in the direction that zero path length position is set,much in the same way as using the tare feature on a scale. The motion isconstrained to reduce or eliminate backlash or recoil as the probe tipand vessel surface are separated. The device capable of these featuresis referred to as a unidirectional sliding mechanism. There are numerousembodiments of the unidirectional sliding mechanism.

In a preferred embodiment, the unidirectional sliding mechanismcomprises a modeled plastic coupling device called a Touhy Borst Adapter(TBA) which contains a silicone rubber or similarly compliant gasketmaterial with a hole in the center of it which is housed by two threadedplastic components which when screwed together compress the internalgasket, thus reducing the diameter of the internal hole creating a sealaround anything within the hole. The amount of sealing and compressioncan be controlled by the changing the length of threaded engagementbetween the two threaded components of the TBA. In a preferredembodiment, the probe tip is inserting through the hole in the TBAgasket and then the TBA is tightened to compress the TBA gasket aroundthe probe tip. The threading is adjusted so the frictional force betweenthe probe tip and the TBA gasket exceeds the weight of the probe tip,thus not allowing the probe tip to fall out of the TBA when heldvertically, but not so tight that the probe tip is unable to slideinside of the gasket. This frictional interaction results in aunidirectional sliding displacement that allows the establishment of thezero path length position.

There are other means and mechanisms by which this can be achieved. Inone embodiment a thin membrane with a hole, a linear slit or twoorthogonal slits enclosed between two blocks contains a hole slightlylarger than the probe tip such that the probe tip can be inserted intothe blocks and the membrane creates the frictional force that allowsdisplacement in one direction.

In another embodiment the coupling mechanism for the probe tip or thesample vessel can comprise a spring loaded tapered sliding coupling thatreleases the probe tip or sample vessel when a force is applied in onedirection, but grips more tightly when the force is released, similar toa spring loaded compression ring.

In another embodiment the coupling mechanism for the probe tip of thesample vessel can comprise a spring loaded ratchet mechanism whichdisplaces a toothed slide which locks in place when displaced in onedirection, but would require a release button to allow unloading ormotion in the opposite direction.

In each of the embodiments of the unidirectional sliding mechanism thezero path length position is set passively, meaning the user does notneed to interact with the device other than driving the motion of thesystem to achieve the physical contact condition. There are otherembodiments that require intervention of the user, which may be utilizedfor long path length and flow versions of the instruments of the presentinvention. In one embodiment, the probe tip coupling mechanism has asliding coupling. After physical contact is achieved and displacementhas occurred the user will set the displacement by means of a thumbscrew, a set screw, tightening a collect, mechanical clamp, magneticclamp or other means of locking the position of either the probe tip,probe tip coupling mechanism, the sample vessel or the sample vesselholding device.

Detector

Detectors comprise any mechanism capable of converting energy fromdetected light into signals that may be processed by the device.Suitable detectors include photomultiplier tubes, photodiodes, avalanchephotodiodes, charge-coupled devices (CCD), and intensified CCDs, amongothers. Depending on the detector, light source, and assay mode suchdetectors may be used in a variety of detection modes including but notlimited to discrete, analog, point or imaging modes. Detectors can usedto measure absorbance, photoluminescence and scattering. The devices ofthe present invention may use one or more detectors although in apreferred embodiment a single detector is used. In a preferredembodiment a photomultiplier tube is used as the detector. The detectorsof the instrument of the present invention can either be integrated tothe instrument of can be located remotely by operably linking thedetector to a light delivery device that can carry the electromagneticradiation the travels through the sample to the detector. The lightdelivery device can be fused silica, glass, plastic or any transmissiblematerial appropriate for the wavelength range of the electromagneticsource and detector. The light delivery device may be comprised of asingle fiber or of multiple fibers and these fibers can be of differentdiameters depending on the utilization of the instrument. The fibers canbe of almost any diameter but in most embodiments the fiber diameter isin the range of from about 0.005 mm to about 20.0 mm.

One preferred embodiment of the instruments of the present invention hasthe optics of the system oriented such that the probe tip is on “top”and the detector is on the “bottom” (FIG. 4). In this verticalorientation the sample vessel is above the detector and the probe tipcan move up and down, into and out of the sample vessel such that thelight form the probe tip moves through the sample within the samplevessel and impinges on the detector below. Other orientations arepossible such as in a flow-cell system where the detector and probe tipmay be in a substantially horizontal orientation (FIG. 5) and the sampleflows between the detector and the probe. Regardless of the absolutespatial orientation or the probe and detector, the probe tip and surfaceof the detector should be substantially perpendicular relative to oneanother.

Software

The control software will adapt the devices behavior based upon variouscriteria such as but not limited to wavelength, path length, dataacquisition modes (for both wavelength/path length), kinetics,triggers/targets, discrete path length/wavelength bands to providedifferent dynamic ranges/resolutions for different areas of thespectrum, cross sectional plot to create abs/path length curves,regression algorithms and slope determination, concentrationdetermination from slope values, extinction coefficient determination,base line correction, and scatter correction. FIG. 1 is a flow diagramof an embodiment of the software scheme of the present invention. Thesoftware is configured to provide scanning or discrete wavelength readoptions, signal averaging times, wavelength interval, scanning ordiscrete path length read options, data processing option such as baseline correction, scatter correction, real-time wavelength cross-section,threshold options (such as wavelength, path length, absorbance, slope,intercept, coefficient of determination, etc.) an kinetic/continuousmeasurement options. FIGS. 2A and 2B are flow diagrams of one embodimentof the data acquisition of the variable path length instrument software.FIG. 3 is a flow diagram of one embodiment of the data acquisition ofreal-time data collection that can be integrated into the dataacquisition program.

FIG. 4A is a schematic of one embodiment of the instruments of thepresent invention. The motor (1) drives the stage (4) on which thesample vessel (3) sits. The fiber tip probe (2) is fixed with respect tothe motor such that as the stage moves up and down the probe distance tothe sample vessel is increase or decreased respectively. Beneath thestage is the detector (5) which receives electromagnetic radiation fromthe probe tip once it has passed through the sample. FIG. 4B is aschematic of one embodiment of the probe tip assembly.

FIG. 5 is a schematic of a flow-through device which may serve as asample vessel in the instruments of the present invention. Theflow-through device comprises a flow cell body (8) that permits the flowof a sample solution into and out of the flow cell device. The flow cellbody (8) has at least one window (7) that is transparent toelectromagnetic radiation in the range of electromagnetic sourcetypically 200-1100 nm. The window can be made from various materials butfor ultraviolet applications quartz, cyclo olefin polymer (COP), cycloolefin copolymer (COC), polystyrene (PS) or polymethyl methacrylate PMMAmay be required. The window may be of different sizes and shapes so longas the electromagnetic radiation can pass through the window and strikethe detector (5). The flow cell body also comprises a port through whichthe probe tip may pass. This port is sealed with a dynamic seal (9) suchthat the probe tip can pass through the port without sample solutionleaking from the flow-through device. Such seals include FlexiSeal Rodand Piston Seals available from Parker Hannifin Corporation EPSDivision, West Salt Lake City, Utah. In the diagram there is a singlepathway for the sample solution to flow coming in the inlet port andexiting the outlet port. Alternative embodiments may include multiplepathways and multiple inlet and outlet ports. In the embodiment of theflow cell device in FIG. 5, the probe tip moves substantiallyperpendicular to the flow of the sample solution and is substantiallyperpendicular to the detector.

In one embodiment of the methods of the present invention multipleabsorbance measurements may be taken at multiple path lengths withoutaccurately knowing what the path length distance is. The prior art isreplete with methods teaching how to accurately determine the pathlength in an absorbance reading so that an accurate determination of theconcentration of the sample can be made. In this embodiment of thepresent invention multiple absorbance measurements made at differentpath lengths enables an accurate calculation of the concentration basedupon the instrument's ability to calculate a regression line from theabsorbance and path length information. The slope of the regression linecan then be used to calculate the concentration of the sample. Each pathlength need not be accurately known due to the fact that the softwareused to calculate the regression line can be programmed to select themost accurate line from the data set presented. The number of datapoints taken in these methods tends to “smooth out” any perturbations inthe path length or absorbance reading such that regression lines withvery high R² values can be obtained. In the methods of the presentinvention R² values of at least 0.99999 have been achieved. Obviouslythe higher the R² value the more accurate the slope which results in ahighly accurate determination of the concentration of the sample. Any R²value between 0 and 0.99999 is achievable in the instruments and methodsof the present invention, however in preferred embodiments of themethods of the present invention the R² value exceeds 0.95000 and inmore preferred embodiments the R² will exceed 0.99500. In a preferredembodiment of the present invention the R² value is between about0.95000 and about 0.99999. Other preferred embodiments include R² valuesbetween about 0.99500 and about 0.99999 and about 0.99990 and about0.99999. While R² is a preferred measure of goodness-of-fit for thelinear regression any other mathematic expression that measuresgoodness-of-fit can be utilized in the methods of the present invention.

The instruments and methods of the present invention allow the user tooptimize the collection of data by selecting a pre-determined parametersuch as absorbance. The user can define, for example, an absorbance of1.0 and have the instrument search for other parameters (such aswavelength or path length) at which the absorbance of the sample is 1.0.This feature enables the user to define the parameters for theexperiment without having to make multiple dilutions or constantlychange the parameters of the instrument manually. The software of thepresent invention also permits the user to define an expected R² valueso that the level of accuracy for the outcome can be defined prior tothe data acquisition.

The instruments and methods of the present invention permit thecollection of a variety of data sets including three dimension data setsthat include measurement of absorbance, path length and wavelength. Thesoftware enables the user to generate three dimensional graphs of thesedata sets. Furthermore, the instruments and methods of the presentinvention provide for the collection of real-time data.

The instruments and methods of the present invention enable thecalculation of the extinction coefficient of a particular sample atdifferent wavelengths. The extinction coefficient, also known asabsorptivity, is the absorbance of a solution per unit path length andconcentration at a given wavelength. If the extinction coefficient for agiven sample is known at a first wavelength (ε₁) one can calculate theextinction coefficient at a second wavelength (ε₂). This is done bymeasuring the ratio of the absorbance/path length at the firstwavelength (A/l)₁ to the absorbance/path length at a second wavelength(A/l)₂ and equating this ratio to the ratios of the extinctioncoefficients: (A/l)₁/(A/l)₂=ε₁/ε₂.

The instruments and methods of the present invention also enable theuser to measure the components in a complex mixture at the same time aslong as the wavelengths that identify the multiple components in thesample can be separated. For example, a conventional spectrophotometerwould not in a single experiment be able to determine the concentrationof a sample where there are two components A, which is highlyconcentrated and absorbs predominantly at 300 nm and B which is quitedilute and absorbs at 600 nm. In a conventional spectrophotometer themeasurement of the absorbance due to component B would preclude themeasurement of the absorbance of component A as the concentration of Ais high enough as to swamp the detector. The original sample would needto be diluted to determine component A, and in doing so component Bwould not produce enough signal to permit its concentration to bemeasured. In a conventional spectrophotometer the concentration of thecomponents A and B cannot be measured simultaneously. In the presentinvention the path length can be altered so that both the concentrationof components A and B can be determined together. Obviously, as long asthere are peaks which uniquely identify a component within a sample themethods of the present invention can measure the concentration of thecomponents of very complex samples. Additionally because the instrumentis capable of generating data in real-time, the interaction ofcomponents within the sample can be monitored to produce kinetic data orany data for which a time course is required.

A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration.

EXAMPLES Example 1 Measurement of concentration of Camphor SulphonicAcid

Camphor sulphonic acid (CSA) ((1S)-(+)-10 camphor sulfonic acid, AldrichC2107-5G) is commonly used to check the calibration of circulardichroism instruments. It has a well defined absorbance peak at 285 nmwith accepted absorbance 0.1486A at 1 cm pathlength and 1 mg/mL. A stockCSA solution was prepared from 1.023 g CSA powder dissolved in 20 mL ofdistilled water to produce a solution of concentration of 51.15 mg/mL(0.2202M). This solution has a calculated absorbance 7.6001 Abs at 1 cmpath length. A second CSA solution was prepared by diluting the stockCSA solution: 4.9 mL of stock was added to 245.1 mL of distilled waterfor a 250 mL total volume. This solution was filtered through 0.2μnalgene filter. The concentration of the diluted solution is 1.00254mg/mL (0.0043M). In FIG. 6 the spectra of both stock and diluted CSAsolutions are shown. The spectra were taken at 1 mm and 10 mm pathlength by Cary400 (standard spectrophotometer) and one embodiment of thepresent invention (SoloVPE). In the case of the Cary 400 the stock anddiluted CSA solution were transferred into cuvettes of path length 1 mmand 10 mm and placed into the Cary 400 for absorbance measurement. Inthe case of the SoloVPE is the path lengths of 1 mm and 10 mm weredetermined by computer control of the probe. The Spectra from SoloVPEshows highly consistance with the Cary 400. This indicates that the pathlengths defined by SoloVPE computer controlled distance are equivalentto the sizes of cuvette used by Cary 400.

Example 2 Measurement of Concentration of Camphor Sulphonic Acid

Stock CSA solution (as described in Example 1) was measured by anembodiment of the invention (SoloVPE) at 285 nm with path length variedfrom 0.05 mm to 2.0 mm in 0.05 mm increments. Diluted CSA solution (asdescribed in Example 1) was measured by SoloVPE at 285 nm with pathlength varied from 1.0 mm to 10.0 mm in 0.1 mm increments. Theexperiment was repeated using a path length range of from 1 mm to 10 mmin 0.1 mm increments. The resulting regression lines from plots of theabsorbance values versus the path length values are shown in FIGS. 2 and3. These values are compared to a single reading at 285 nm in a Cary 400spectrophotometer taken for the stock and diluted samples of CSAsolution in a 10 mm cuvette. Using slope spectroscopy the sampleconcentration can be obtained from the linear regression curve of theabsorbance vs. pathlength data. FIGS. 7 and 8 are the plots ofabsorbance vs. pathlength data from both stock and diluted solutions,respectively. The instrument (SoloVPE) measured the absorbances of stocksolution with path lengths varied from 0.05 mm to 2.0 mm and dilutedsolution from 1 mm to 10 mm. The slope of linear regression curve forstock CSA solution is 0.756031 with linear correlation coefficientR²=0.99999. The diluted CSA solution data has 0.0145 slope andR²=0.9997. Based on the equation, Absorbance (A)/path length(l)=extinction coefficient (ε)×concentration (c), slope values from theregression (A/l) were used to obtain the solution concentration. In thistest, the concentration of stock solution is 50.88 mg/mL (0.219M) anddiluted solution is 0.976 mg/mL (0.0042M). Compare with concentrationvalues of the sample based on the composition of the samples fromExample 1, the results obtained by the slope regression measurements atmultiple path lengths have −0.53% and −2.6% difference for stock anddiluted solutions respectively.

Example 3 Measurement of Wavelength Peaks at Multiple Path Lengths forPatent Blue

Patent Blue Standard was purchased from GFS Chemical, Inc., Columbus,Ohio. Patent Blue standard has absorbance peaks reported at 310 nm, 412nm, and 639 nm wavelengths. In FIG. 9, the absorbance peaks at 310 nmand 412 nm can be easily identified in these path length scans. Eventhough both peaks can be seen in the plot, 412 nm peaks are alreadyclearly defined at 15 mm path length while 310 nm peaks are noisybetween 10-15 mm path lengths. This indicates that the signals at 310 nmwavelength close to the saturation level of the detector at the pathlengths greater 10 mm. A clear 310 nm peak can be defined at pathlengths greater than 10 mm. The 639 nm absorbance peak is absent inlonger path lengths range and is not seen until the path length isreduced to about 1 mm.

As the path lengths are reduced from 1.5 mm to 0.1 mm, (FIG. 10) thesize of the three absorbance peaks is commensurately reduced. Theabsorbance peaks at 310 nm and 412 nm reached zero absorbance ordetector noise level while the 639 nm absorbance peaks remain measurableand provide meaningful information. The data from FIGS. 9 and 10 werecollected in one run from SoloVPE. For all commercial availablespectrophotometers, one has to take several steps, such as dilutingsamples and changing different sizes of cuvette, to obtain same results.

Example 4 Measurement of Concentrated Bovine Serum Albumin

BSA solution was purchased from Sigma-Aldrich Co., P/N A7284 300 mg/mL.BSA sample has optical absorbance 0.667 Abs at 279 nm for 1 gm/Lconcentration. In this example, the concentration of BSA is 300 mg/mL±10% error according to the data provided by Sigma-Aldrich. Theabsorbance scans of this BSA solution in 10 mm and 1 mm cuvettes fromCary 50 Spectrophotometer are shown in FIG. 11. Both absorbance valuesat 279 nm saturate the detector because of the high concentration of thesolution. FIG. 12 is the absorbance scan of same solution at 200 μm pathlength using an instrument of the present invention. This scandemonstrates that the absorbance value at this small path length(smallest commercially available cuvette) also saturates the detector.FIG. 13 is the spectra of the BSA solution taken by the SoloVPEinstrument at 0.1 mm to 0.01 mm path lengths with 0.005 mm steps. In thetested path lengths range, the absorbance peak at 279 nm wavelength doesnot saturate the detector. Collecting absorbance values at 279 nm ofeach path lengths, a plot of the absorbance vs. path length (FIG. 14)and regression analysis yields a concentration of the BSA solution of330.6 mg/mL.

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the invention as claimed.

We claim:
 1. A method of determining the concentration of a samplecomprising: (a) taking multiple absorbance measurements at multiple pathlengths of the sample; (b) generating a regression line from theabsorbance values and path length such that a slope of the regressionline is obtained; and, (c) determining the concentration of the sampleby dividing the slope of the regression line by the extinctioncoefficient of the sample.
 2. The method of claim 1 wherein the pathlength distance is not accurately known.
 3. The method of claim 1wherein the number of absorbance readings provides an R² value of theregression line of at least 0.95000.
 4. A method of determining theconcentration of two or more components in a sample comprising: (a)taking multiple absorbance measurements at multiple path lengths atmultiple wavelengths of the sample; (b) generating a regression linefrom the absorbance values and path length at each wavelength such thata slope of the regression line is obtained for each wavelength; and, (c)determining the concentration of each of the components of the sample bydividing the slope of the regression for each of the wavelengths line bythe extinction coefficient of the respective component in the sample. 5.An instrument for measuring the scattered light in a sample comprising:(a) a light source linked operably to a probe; (b) a sample vessel thatcan contain the sample; (c) a motor operably linked to the sample vesselsuch that the sample vessel is moved relative to the probe to providevariable path lengths; (d) a probe that can carry electromagneticradiation that is moved relative to the sample vessel by the motor; (e)a universal sliding mechanism; and (f) a detector that can detect lightscattering.